ABSTRACT Malaria is a public health problem of global importance. This devastating disease infects 198 million infections people every year and kills 584,000, mostly young children in sub-Saharan Africa. Emerging resistance to drugs used on a large scale is now one of the greatest obstacles to the control of malaria. As the problem of drug resistance arises at the interface of chemistry and biology, so too must the solution. My goal is to use chemical tools to probe parasite biology in order to develop new solutions to the problem of antimalarial resistance. Using an integrated chemogenomic approach, we have identified the cytoplasmic prolyl tRNA synthetase in Plasmodium falciparum (PfcPRS) as the long-sought biochemical target of halofuginone. Furthermore, we uncovered an unprecedented mechanism of drug-tolerance in the parasite by modulation of proline homeostasis. In this proposal, I seek to understand the molecular basis of the parasite's ability to sense and evolve resistance to halofuginone. I will investigate a non-genetic mechanism of resistance to halofuginone by investigating the primary source of increased intracellular proline in response to halofuginone treatment. I will also explore the underlying mechanisms of aaRS inhibition in the parasite by evaluating the role of the amino acid response in sensing and responding to aaRS inhibition. Insights gained from the proposed experiments could reveal novel targets for chemotherapeutic intervention, prevent and overcome resistance to PfcPRS inhibitors, and potentially identify synergistic combination treatment strategies. Successful execution of this proposal will enable us to delve into the biology of the parasite in order to find a new Achilles' heel to exploit. This will put us on the path to saving the next generation of people from the morbidity and mortality of malaria.